Ionotropic glutamate receptors (iGluRs) are membrane proteins that act as molecular pores and mediate signal transmission at the majority of excitatory synapses in the mammalian nervous system. iGluRs are also found in primitive prokaryotes and eukaryotes suggesting an early evolutionary origin, followed by gene expansion to generate subtypes with diverse functional properties. The 7 gene families of ionotropic glutamate receptors (iGluRs) in humans encode 18 subunits which assemble to form 3 major functional families named after the ligands which were first used to identify iGluR subtypes in the late 1970s: AMPA, kainate and NMDA. Because of their essential role in normal brain function and development, and increasing evidence that dysfunction of iGluR activity mediates multiple neurological and psychiatric diseases, as well as damage during stroke, a substantial effort in the Laboratory of Cellular and Molecular Neurophysiology is directed towards analysis of iGluR function at the molecular level. Atomic resolution structures solved by protein crystallization and X-ray diffraction provide a framework in which to design biochemical and electrophysiological experiments to define the mechanisms underlying ligand recognition, the gating of ion channel activity, and the action of allosteric modulators. This important information will allow the development of subtype selective antagonists and allosteric modulators with novel therapeutic applications and reveal the inner workings of a complicated protein machine which plays a key role in brain function. NMDA RECEPTOR GLYCINE AND GLUTAMATE LIGAND-BINDING DOMAINS The NMDA receptor family of glutamate receptor ion channels are formed by obligate heteromeric assemblies of GluN1, GluN2 and GluN3 subunits. GluN1 and GluN3 bind glycine, whereas GluN2 binds glutamate. Crystal structures of the GluN1 and GluN3A ligand-binding domains (LBDs) in their apo states unexpectedly reveal open and closed cleft conformations, respectively, with water molecules filling the binding pockets. Computed conformational free energy landscapes for GluN1, GluN2A, and GluN3A LBDs reveal that the apo state LBDs sample closed cleft conformations, suggesting that their agonists bind via a conformational selection mechanism. By contrast, free energy landscapes for the AMPA receptor GluA2 LBD suggests binding of glutamate via an induced fit mechanism. Principal component analysis reveals a rich spectrum of hinge bending, rocking, twisting, and sweeping motions that are different for the GluN1, GluN2A, GluN3A, and GluA2 LBDs. This variation highlights the structural complexity of signaling by glutamate receptor ion channels. STRUCTURAL STUDIES ON THE EVOLUTION OF iGLURS AvGluR1, a glutamate receptor ion channel from the primitive eukaryote Adineta vaga , is activated by alanine, cysteine, methionine, and phenylalanine, which produce lectin-sensitive desensitizing responses like those to glutamate, aspartate, and serine. AvGluR1 LBD crystal structures reveal an unusual scheme for binding dissimilar ligands that may be utilized by distantly related odorant/chemosensory receptors. Arginine residues in domain 2 coordinate the gamma-carboxyl group of glutamate, whereas in the alanine, methionine, and serine complexes a chloride ion acts as a surrogate ligand, replacing the gamma-carboxyl group. Removal of Cl_ lowers affinity for these ligands but not for glutamate or aspartate nor for phenylalanine, which occludes the anion binding site and binds with low affinity. AvGluR1 LBD crystal structures and sedimentation analysis also provide insights into the evolutionary link between prokaryotic and eukaryotic iGluRs and reveal features unique to both classes, emphasizing the need for additional structure-based studies on iGluR-ligand interactions. NOVEL ZINC BINDING SITES ON GLUK3 SUBTYPE KAINATE RECEPTORS Kainate receptors (KARs) play a key role in the regulation of synaptic networks. Here, we show that zinc, a cation released at a subset of glutamatergic synapses, potentiates glutamate currents mediated by homomeric and heteromeric KARs containing GluK3 at 10100 micro Molar concentrations, whereas it inhibits other KAR subtypes. Potentiation of GluK3 currents is mainly due to reduced desensitization, as shown by kinetic analysis and desensitization mutants. Crystallographic and mutation analyses revealed that a specific zinc binding site is formed at the base of the ligand binding domain (LBD) dimer interface by a GluK3-specific aspartate (Asp759), together with two conserved residues, His762 and Asp730, the latter located on the partner subunit. In addition, we propose that tetrameric GluK2/GluK3 receptors are likely assembled as pairs of heterodimeric LBDs. Therefore, zinc binding stabilizes the labile GluK3 dimer interface, slows desensitization, and potentiates currents, providing a mechanism for KAR potentiation at glutamatergic synapses. CRYO EM STRUCTURES OF FULL LENGTH iGLURS Insight into molecular mechanisms underlying glutamate receptor gating is limited by lack of structural information for receptors trapped in different conformational states.Here,we report the use of single-particle cryoelectron tomography to determine the structures, at 21 Angstrom resolution, of full-lengthGluK2 kainate receptors trapped in antagonist-bound restingand agonist-bound desensitized states. The resting state, stabilized by the competitive antagonist LY466195, closely resembles the crystal structure of the AMPA receptor GluA2,withwell-resolved proximal and distal subunits exhibiting cross-over between the twofold symmetric amino terminal domain and a twofold symmetric ligand binding domain (LBD) dimer of dimers assembly. In the desensitized state, the LBD undergoes a major rearrangement, resulting in a separation of the four subunits by 25 Angstroms. However, the amino terminal domain, transmembrane, and cytoplasmic regions of the receptor have similar conformations in the resting and desensitized states. The LBD rearrangement was not anticipated in prior models based on crystal structures for soluble LBD dimer assemblies, and we speculate that subunit separation allows a better match to the fourfold symmetric ion channel domain. From fits of the amino terminal domain and LBD domains into the density map of the desensitized state we have derived a structural model for differences in quaternary conformation between the resting and desensitized states.